Improved Methods of Cell Selection for Bone Marrow Transplant Alternatives

The NHLBI Hematology Branch Stem Cell Transplantation program is exploring ways to make
allogeneic transplantation safer and more widely applicable. Prior Hematology Branch
transplant protocols have evaluated the strategy of using T cell depleted marrow transplants
followed by delayed lymphocyte add-back to control or prevent GVHD while conserving useful
donor immune function against residual leukemia and infectious agents. Over the past ten
years, a number of increasingly efficient methods have been used to deplete T cells but
retain stem cells, and we have shown the safety and utility of the delayed T cell add-back
approach. We have also found a positive relationship between administration of higher CD34+
cell doses and outcome. Investigation of highly purified grafts with add-back of specific T
cell populations is ongoing, and the ability to test new purification approaches and devices
on clinical-scale PBSC products is critical to the continued development of new
transplantation approaches in our program. This requires testing the approaches on G-CSF
mobilized PBSCs collected by apheresis from healthy donors, since this is the cell source
that will be used in all clinical allogeneic transplantation protocols in our program.

Therefore, the primary intent of this protocol is to provide a mechanism for mobilizing,
collecting, storing, and analyzing G-CSF mobilized apheresis samples from healthy volunteers.
Cells will be used to develop a method of processing the cells that are collected after
stimulation with G-CSF, by removing the lymphocytes, which can mediate GVHD while retaining
the stem cells which are necessary for hematopoietic reconstitution. At the same time we will
study whether G-CSF administration has an effect on the lymphocyte, function which may
influence the immune reactions occurring in allogeneic bone marrow transplantation.
Furthermore the CD34+ cells collected will be a valuable resource for experimental studies of
lymphocyte-stem cell interactions in our laboratory.

- INCLUSION CRITERIA:

Healthy healthy individual aged between 18 and 60 years.

No active infection or history of recurrent infection.

Normal renal function: creatinine less than 1.5 mg/dl, proteinuria less than 1+.

Normal liver function: bilirubin less than 1.5 mg/dl, transaminase less than 1.5- fold
upper limit of normal

Normal blood count: WBC 3000 to 10,000/mm(3), granulocytes greater than 1500/mm(3),
platelets greater than 150,000/mm(3), hemoglobin greater than 12.5 g/dl, MCV and MCHC
normal.

Normal cardiovascular function, no history of chest pain, myocardial infarction, peripheral
vascular disease, transient ischemic attack, or stroke.

Healthy female subjects of childbearing age should have a negative serum pregnancy test
within one week of beginning G-CSF administration.

Female subjects should not be lactating.

Subject must be eligible for normal blood donation. He or she must be tested negative for
syphilis (RPR), hepatitis B and C (HBsAg, Anti HBc, Anti HCV), HIV and HTLV 1.

Subject must be able to comprehend the investigational nature of the study and provide
informed consent to participate in the protocol.

Antecubital veins must be adequate for peripheral access during apheresis. Potential
participants must be screened by an apheresis nurse to check venous access before protocol
entry.

EXCLUSION CRITERIA:

Active viral, bacterial, fungal or parasite infection.

Female with positive pregnancy test or lactating.

History of autoimmune disease such as rheumatoid arthritis, systemic lupus erythematosus.

History of cancer excluding squamous carcinoma of the skin.

History of any hematologic disorders.

History of cardiovascular disease or related symptoms such as chest pain, shortness of
breath, history of cerebrovascular disease.

Any positive serum screening test as listed in eligibility.

Allergy to G-CSF or bacterial E coli products.

Administration of NSAID within 10 days of starting protocol.

History of G-CSF administration and leukapheresis within past 3 months.